1. Field of the Invention
The present invention relates generally to the field of immunology. More specifically, the present invention involves identification of dominant CD8 T cell epitopes in the Human Papilloma virus (HPV) proteins and its use in treating cancer such as cervical cancer.
2. Description of the Related Art
Cervical cancer is the second most common malignancy among women worldwide (1) with 400,000 new cases annually (2). Annually 12,000 to 14,000 new cases of squamous cell cancer of the cervix are reported in the United States (3), resulting in about 3,500 deaths per year. High-risk Human Papilloma virus, the most commonly HPV16, is the major cause of cervical cancer (4-5). Among the over one hundred different types of Human Papilloma virus, at least 15 are strongly associated with invasive squamous cell cancer of the cervix (6). HPV16 is the one most commonly found associated with this cancer (7-8).
Human Papilloma virus infection is also associated with the precursor lesion of cervical cancer, squamous intraepithelial lesion (7-12). While most low-grade squamous intraepithelial lesions prospectively regress spontaneously (13-14), some progress to high-grade squamous intraepithelial lesions. These high-grade lesions, in particular, cervical intraepithelial neoplasia-3 is associated with a high rate progression to invasive cervical cancer (15-16).
Two early gene products, E6 and E7, mediate transformation to a malignant phenotype by Human Papilloma virus. Both of these viral proteins have been shown to interact with the products of cellular human tumor suppressor genes. The E6 protein can bind and promote degradation of cell-encoded p53, while the E7 protein interacts with the retinoblastoma susceptibility gene product. Constitutive expression of HPV E6/E7 proteins is required for the maintenance of a malignant phenotype of cervical cancer (5, 17). Moreover, HPV16 E6 and E7 proteins contain many antigenic epitopes and are foreign viral antigens. These proteins may, therefore, represent targets of antigen-specific immunotherapeutic strategies for the prevention and treatment of cervical cancer.
Cell-mediated immunity plays an important role in controlling Human Papilloma virus infection and Human Papilloma virus-associated diseases. CD4 T cells are important in the development of anti-tumor responses (18-21). It is believed that the effectiveness of these CD4 T cells lies in their ability to deliver help for priming and maintaining CD8 cytotoxic T lymphocytes, which are thought to serve as the dominant effector cells in tumor elimination. Immunohistochemical analyses of squamous intraepithelial lesions and cervical cancer specimens have demonstrated the presence of activated cytotoxic T lymphocytes in lesions (22). The CD4 T cells activate cytotoxic T lymphocytes by producing T helper 1 cytokines (23) and by providing activation signals for priming of tumor-specific cytotoxic T lymphocytes to professional antigen presenting cells (24-27). CD8-positive cytotoxic T lymphocytes recognize foreign peptides that are 8 to 11 amino acids in length and bound to and presented by Human Leukocyte Antigen class I molecules. These peptides are called T cell epitopes.
A study identified epitopes of HPV16 E6 and E7 proteins by using overlapping peptides of these proteins to stimulate peripheral blood mononuclear cells from a healthy donor and binding assays to find candidate epitopes (28). This approach enabled the identification of Human Leukocyte Antigen-B18 epitopes, E6 80-88 (ISEYRHYCY; SEQ ID NO: 24) and E7 44-52 (QAEPDRAHY: SEQ ID NO: 28). It was also shown that E6 80-88 was a naturally processed epitope that could be recognized by T cells from a patient with high-grade squamous intraepithelial lesion. Although the binding of the peptide to the Human Leukocyte Antigen molecule was demonstrated, the strength of the T cell response to these antigenic epitopes compared with other T cell epitopes was not assessed, and it was not clear whether this peptide had a protective effect.
A study using stimulated peripheral blood mononuclear cells from cervical cancer patients with an Human Leukocyte Antigen-A2-restricted HPV16 E7 peptide (E7 11-20) showed that cytotoxic T lymphocytes were capable of lysing Human Leukocyte Antigen-matched HPV16 E7 11-20-pulsed targets in two of three patients (29). Further, another group identified HPV-specific cytotoxic T lymphocytes in lymph nodes and tumors of cervical cancer patients (30). In previous work examining cytotoxic T lymphocyte responses to HPV16 in HPV16-infected women (no squamous intraepithelial lesion), cytotoxic T lymphocyte responses to the HPV16 E6 protein, but not to the E7 protein, were significantly associated with the clearance of HPV16 infection (31).
These observations have demonstrated HPV16 E6- and/or E7-specific cytotoxic T lymphocytes in women with and without squamous intraepithelial lesion and in women with cervical cancer. Efforts have been made to define the viral epitopes inducing the Human Papilloma virus-specific cytotoxic T lymphocyte that are responsible for the clearance of virus-infected and virus-transformed cells. Using the same approach as was taken for HPV16, Human Leukocyte Antigen-A2.1 binding synthetic peptides of HPV18 E6 protein were identified (32). Some of these binding peptides were also shown to be antigenic by demonstrating in vitro cytotoxicity.
High-risk human Papilloma virus peptide antigens for CD8 T lymphocytes have been shown to be antigenic in human experimental systems by demonstrating peptide-specific cytotoxicity or interferon-γ secretion. Except for the Human Leukocyte Antigen-B18-restricted epitopes identified by Bourgault Villada et al., all were pre-selected for the given Human Leukocyte Antigen types. None of the antigenic epitopes were identified based on the magnitude of T cell response regardless of the restricting Human Leukocyte Antigen molecules.
Memory T cells play an important role in maintaining long-term immunity to previously encountered pathogens or tumor antigens. They may proliferate, and rapidly acquire effector functions to kill virus-infected cells or tumor cells, and secrete cytokines that inhibit replication of the pathogen after re-stimulation with re-exposure to antigen (33). Antigen presenting cells, which may transfer peripheral antigenic signals to the lymphoid organs, play a crucial role in the induction of antigen-specific T cell immunity responses to Human Papilloma virus infection and Human Papilloma virus-associated tumors. Dendritic cells as professional antigen presenting cells express high level of major histocompatibility complex and co-stimulatory molecules. Insufficient or improper activation of dendritic cells, caused by lack of pro-inflammatory signal, leading to antigen presentation not in an appropriate co-stimulatory context is one reason for the failure of antitumor immunity. Vaccination with autologous, tumor antigen loaded properly activated dendritic cells in vitro present promising immunotherapy modality for tumors. With the development of techniques for dendritic cell isolation, antigen loading and maturation, dendritic cell-based vaccines has progressed in recent decade (34-35).
Thus, the prior art is deficient in peptide antigens, derived from the Human Papilloma virus E6 protein that have been identified based on the T cell responses, to be used as sources of antigens for therapeutic vaccines or for dendritic cell immunotherapy to treat cervical cancers. The present invention fulfills this long-standing need and desire in the art.